Accuracy in translation of the genetic code into proteins depends upon correct recognition of their cognate messenger RNA (mRNA) codons by transfer RNA (tRNA) anticodons. Within tRNAs, incorporation of posttranscriptionally modified bases is common; however, the roles of these chemical modifications are poorly understood. In human tRNA^Lys,3_UUU three modified bases are present—2-methylthio-6-threonylcarbamoyladenosine at position 37 (ms²t⁶A37), 5-methyoxycarbonylmethyl-2-thiouridine at position 34 (mcm⁵s²U34), and pseudouridine (ψ) at position 39—two of which, ms²t⁶A37 and mcm⁵s²U34, are required to achieve wild-type binding activity of wild-type human tRNA^Lys,3_UUU (Yarian et al., Biochemistry, 2000, 39, 13390). To elucidate the function of each modified base in the human tRNA^Lys,3_UUU anticodon stem loop, molecular dynamics simulations of eight tRNA anticodon stem loops with different combinations of nonstandard bases were performed. RESP charges for nonstandard bases ms²t⁶A, mcm⁵s²U, and ψ were derived and incorporated into the Cornell et al. (J. Am. Chem. Soc., 1995, 117, 5179) force field. From these simulations it was found that models with ms²t⁶A37 retained the stair-stepped conformation of the anticodon bases and prevented U36, the anticodon reading the first codon, from flipping out into solution.